The double stranded RNA (dsRNA)-activated protein kinase PKR, also referred to as P1/elF2 kinase, dsRNA-activated inhibitor (DAI or dsl), and p68 (human) or p65 (murine) kinase, is a ubiquitously expressed serine/threonine protein kinase that was initially identified as an interferon (IFN)-induced protein and characterized as a translational inhibitor in an antiviral pathway regulated by IFNs (reviewed in Stark, G. R., et al., Ann. Rev. Biochem. 67:227-264, (1998)).
PKR has been shown to play a variety of important roles by regulating translation, transcription and signal transduction pathways through its ability to phosphorylate various intracellular proteins. One well characterized role of PKR is the phosphorylation of eukaryotic initiation factor 2 alpha-subunit (elF-2alpha), which once phosphorylated, ultimately leads to inhibition of cellular and viral protein synthesis Hershey, J. W. B., Ann. Rev. Biochem. 60:717-755, (1991)).
In addition to mediating the antiviral activities of IFN-alphas, PKR has more recently been implicated in other aspects of cellular function including normal growth control, induction of apoptosis and signal transduction (Williams, B. R. G., Biochem. Soc, Trans. 25:509-513, (1997); Yeug, M. C., et al., Proc. Natl. Acad. Sci., USA 93:12451-12455, (1996)). Its role as a signal transducer has been established for dsRNA, lipopolysaccharide (LPS) and the proinflammatory cytokines, tumor necrosis factor (TNF), interleukin-1beta and IFN-gamma. PKR has been shown to mediate signal transduction through activation of important transcription factors such as nuclear factor kappa B (NF-κB), activating transcription factor-2 (ATF-2), signal transducer and activator of transcription-1 (STAT-1) and interferon regulatory factor-1 (IRF-1) Williams B. R. G., Oncopene 18:6112-6120, (1999)).
When PKR is overexpressed in cells and subsequently activated by dsRNA, it results in increased production of the antiviral cytokines IFN-alpha and IFN-beta, the proinflammatory cytokines TNF-beta and IL-6 and the proinflammatory chemokine IL-8. (WO 97/08324, Lau A. S.). An additional important and relevant recent finding was that LPS induction of the proinflammatory cytokines IL-6 and IL-12 was defective in PKR-null cells and impaired in PKR-null mice (Goh, et al., 2000). Thus, PKR is a key player in the cellular response to various stress signals that culminate in the synthesis of pro-inflammatory cytokines.
Activation of PKR has dualistic effects on cellular homeostasis. It results in the initiation of protein synthesis that inhibits pathogen/cancer growth, and concomitantly activates the transcription of proinflammatory cytokines and other genes. Taken together, these events limit pathogen invasion and favor the propagation of inflammatory responses to enhance immune defense.
Cytokines are soluble proteins produced during the activation of innate and acquired immune response to a variety of tissue insults including infection, cancer and autoimmunity. Cytokines have pleiotropic effects, acting on many cell types to modulate the host's immune response and are the principal means for intercellular communication during an insult. Cytokines initiate the inflammatory response and define the magnitude and nature of the immune response. However, overproduction of proinflammatory cytokines can result in extensive damage to host tissues, leading to morbid states, and in severe cases, eventual mortality of the host. Examples of inflammatory diseases include rheumatoid arthritis, inflammatory bowel disease, malignancy-associated cachexia and septic shock Abbas, A. K., et al.,Eds., Cellular and Molecular Immunology, 4th edition, WB Saunders Co., 246-415, 2000)). Other diseases where inflammatory cytokines may play a role include cerebral stroke, congestive heart failure and various inflammatory lung diseases such as adult respiratory distress syndrome Barone, F. C. and Feuerstein G. Z., J. Cereb. Blood Flow Meta 19:819-834, (1999); Torre-Amione, G., et al., Drugs 59:745-751, (2000); Christman, J. W., et al., Chest 117:1482-1487, (2000)).
Induction of many proinflammatory cytokines involves the activation of nuclear transcription factors, and NE-κB is a prototype of these regulatory factors (Baldwin, A. S., Ann. Rev. Immunol. 14:649-681, (1996)). Binding sites for NF-κB are present in the promoter regions of cytokine genes important in inducing the inflammatory responses associated with the fore-mentioned diseases (Abraham, E., Critical Care Med 28:100-104, (2000)). Therefore, a common mechanism of many anti-inflammatory drugs is to suppress the production of one or more these proinflammatory cytokines through the suppression of signaling pathways such as those involving NE-κB (Handel, M. L., et al., Clin Exp Pharmacol Physiol. 27:139-144, (2000)).
Drug discovery in the area of anti-inflammatory compounds has the potential to identify novel compounds that are more effective and/or better tolerated than existing anti-inflammatory compounds. Another important criterion for selection of a target is that suppression of such target does not induce immediate or long term deleterious effects or toxicity to the host. Recent reports on PKR-knockout mice showed that deletion of the PKR gene does not cause inherent toxicity or compromise the survival of the said animals, with the exception of diminished antiviral response. Yet, embryonic fibroblast cells derived from these animals have much less proinflammatory response following endotoxin stimulation (Williams, 1999, Goh, et al., 2000). Thus targeting PKR suppression should be a safe approach to ameliorate inflammation without serious untoward effects.
PKR is an upstream regulator of NF-κB activation and proinflammatory cytokine synthesis and therefore is useful as a new target for the development of potentially novel anti-inflammatory drugs. Ideally, the drug discovery approaches that are used can be adapted to high-throughput screening (HTS), allowing large numbers of compounds, e.g., combinatorial library compounds, to be screened in a short time, and at a relatively low cost. To date, there is a need for a cell system for identifying candidate anti-inflammatory compounds by high throughput screening or other facilitated screening methods, in particular, a system to screen candidates to suppress PKR activation and its consequent induction of undesirable cytokines. The present invention addresses this need.